1. Field of the Invention
This invention relates to the field of identification or analysis of compounds. More particularly, the invention relates to enhancing the confidence index in detecting the presence and/or amount of an analyte and to the derivatization of organic compounds carried out in conjunction with the analysis or determination of various organic compounds. The present invention is described hereinbelow with emphasis on the analysis of organic compounds that are drugs of abuse and on the use of chromatography with mass spectroscopy in the analysis.
The clinical diagnostic field has seen a broad expansion in recent years, both as to the variety of materials of interest that may be readily and accurately determined, as well as the methods for the determination. Convenient, reliable and non-hazardous means for detecting the presence of low concentrations of materials in liquids is desired. In clinical chemistry these materials may be present in body fluids in concentrations below 10xe2x88x9212 molar. The difficulty of detecting the presence of these materials in low concentrations, as well as the confidence in their detection, are enhanced by the relatively small sample sizes that can be utilized.
Over the last decade, testing for drugs of abuse has become commonplace. This testing is not only for the monitoring of criminal offenders and drug addicts, but employers also use it for the screening of workers. In general, samples of interest are first tested by less expensive means such as immunoassays and the like. Such immunoassays include those based on detection employing various labels such as enzymes, fluorescent compounds, radioactive materials and so forth. Once a sample has been identified as positive for the presence of an analyte, it is often necessary or required to perform a confirmation test, which may be an immunoassay that differs from that used in the initial testing. In the area of testing for drugs of abuse, a confirmatory test may involve much more sophisticated and expensive technology such as gas chromatography and mass spectrometry or combinations thereof.
In recent years, techniques have been developed for the analysis or determination of organic compounds present in extremely small quantities or at very low concentrations. For example, by combining chromatographic techniques such as gas chromatography with various detection means such as mass spectrometry, sensitivity in the detection of analytes is enhanced.
The use of mass spectrometry for the identification of compounds and determination of their molecular structure is well known in the art. In one form of a mass spectrometer, a sample gas is partially ionized by electron impact or other means in an ion source. For each compound in the sample, a set of fragment ions are typically formed, each one having a particular mass to charge ratio, which is usually referred to as xe2x80x9cmass.xe2x80x9d
The ions are separated by electric, magnetic or combined fields (in a mass analyzer) into different species according to their respective masses. In the usual arrangement of the mass analyzer, ions of one mass at a time are transmitted to a suitable detector for measurement and/or recording. Usually, the mass analyzer controls are manipulated so that the mass to charge ratio values are repeatedly and continuously swept over a selected mass range. A plot or tabulation of ion current or intensity versus mass to charge ratio is referred to as a mass spectrum and is the basic data output from a mass spectrometer.
By combining the techniques of gas chromatography with electron capture detection or mass spectrometry operating in the negative ion chemical ionization (NCI) mode, organic chemicals present in solution at concentrations of micrograms per ml or less can be analyzed. Such techniques are extremely useful for the analysis of aqueous fluids obtained from living organisms, such as plasma, serum, urine, etc., from man and other animals and liquid media from microorganisms.
One characteristic of such highly sensitive techniques is that they require the presence of certain specific groups in the molecule, for example, chromophores, fluorophores or electrophores. For instance, electron capture detection requires the presence of an electrophore (a group capable of capturing electrons), e.g., a group containing halogen atoms covalently bound to carbon. Unfortunately, many organic compounds of interest for analysis, particularly those from biological fluids mentioned above, do not possess such groups and must be converted to suitable derivatives as a preliminary step. In some cases it is also necessary to convert unstable compounds to stable derivatives.
There is a general reservation in the minds of at least some of those skilled artisans in using NCI for confirmation of the presence of a certain organic compound when there is limited fragmentation. For example, in the area of analyses for drugs of abuse, the use of NCI for confirmation of a positive test result from another testing method such as, for example, immunoassays and the like has not been widely adopted. In the latter situation there is only a primary molecular anion as the most intense ion in NCI. Those skilled in the art have focused their efforts on obtaining a single peak by purifying components and have achieved high intensity of signal. However, although signals of high intensity have been achieved, there is not enough mass information to provide a level of comfort amongst those skilled in the art, particularly in the area of confirmation of positive results in testing for drugs of abuse.
2. Brief Description of Related Art
U.S. Pat. No. 4,224,031 (Mee, et al.) discusses the chemical ionization mass spectrometric analysis of physiologically active compounds.
U.S. Pat. No. 4,990,458 (Rosenfeld) discloses the derivatization of organic compounds during their analysis or determination.
Use of mixed anhydrides for the determination of terfenadine in dosage forms and spiked human plasma is disclosed by Al-Majed, et al., in Journal of Pharmaceutical and Biomedical Analysis (2000) 23:281-289.
One embodiment of the present invention is a method for enhancing the confidence in detecting the presence of an analyte in a sample suspected of containing the analyte. In the method, a combination of at least two predetermined derivatives of the analyte is subjected to chromatographic separation. The predetermined derivatives exhibit different retention times as a result of the chromatographic separation. Then, the retention times of the derivatives are determined. The retention times are related to the presence of the analyte in the sample. This is a two-dimensional approach that relates the retention times as indicated by the detector signal to the presence of the compound.
Another embodiment of the present invention is a method for detecting the presence and/or amount of an analyte in a sample suspected of containing the analyte. A combination comprising at least two predetermined derivatives of the analyte is subjected to chromatographic separation to separate the derivatives, which are subjected to ionization to form ions as they exit from the chromatograph. A response from each of the ions is detected. The retention times of the ions and the ratios of the intensities of the responses are determined. The retention times and the ratios are related to the presence and/or amount of the analyte in the sample.
Another embodiment of the present invention is a method for detecting the presence and/or amount of a drug in a sample suspected of containing the drug. The sample is combined with at least two predetermined derivatizing agents to form at least two derivatives of the analyte. The derivatives are subjected to gas chromatographic separation to separate the derivatives. The separated derivatives are subjected to chemical ionization to form ions of the derivatives. The ions are subjected to mass analysis and a response from each is detected. The retention times of the ions and the ratios of the intensities of the responses are determined and the retention times and the ratios are related to the presence and/or amount of the drug in the sample.
Another embodiment of the present invention is a method for detecting the presence and/or amount of a drug of abuse in a sample suspected of containing the drug of abuse. The sample is combined with at least two predetermined derivatizing agents and then subjected to conditions under which derivatives of the analyte are formed. The derivatives are subjected to gas chromatographic separation to separate the derivatives and the separated derivatives are subjected to negative ion chemical ionization to form negative ions of the derivatives. The ions are subjected to mass analysis and a response from the ions is detected. The retention times of the ions and the ratios of the intensities of the responses are determined. The retention times and the ratios are related to the presence and/or amount of the drug in the sample.